A series of whole animal studies have clearly demonstrated selective brain neurodegeneration in a binge-type alcohol rodent model. Pyramidal cells of layer III of the entorhinal cortex (ENT) and hippocampal (HIPP) dentate gyrus granule cells are particularly vulnerable to the cytotoxic consequences of binge-type alcohol exposure. The main purpose of this project was to establish an experimental model to investigate alcohol-induced neurodegeneration in vitro that would complement our in vivo studies. Organotypic rat brain slices provide a culture system that preserves the main morphological and neurochemical features of brain tissue. Furthermore, organotypic slice cultures retain their native circuitry, cell types and synaptic density with conservation of main afferents. Ongoing studies have revealed that the ENT-HIPP explants are optimal for study at 2-3 weeks post harvest when slices are obtained from 8-day old neonates. The ENT-HIPP explants were treated with ethanol (0.2-0.6%) over 6 days using several different alcohol exposure paradigms. Alcohol-induced neuronal injury and/or death were assessed by the extent of uptake of propidium iodide(PI,cytotoxic marker) and the amount of lactate dehydrogenase(LDH), another cytotoxic marker, in the explant medium. Treatment of ENT-HIPP slices cultured in serum-free media containing B27 neuronal supplement with 0.2-0.4% alcohol for 2-6 days resulted in dose- and time-related increases in PI uptake and LDH leakage. The presence of classical antioxidants (glutathione, vitamin E,catalase and superoxide dismutase) did not lessen the alcohol induced cytotoxicity. Furthermore, agents that were found to be cytoprotective in vivo in the presence of alcohol, such as furosemide and cannabadiol, were not protective in vitro. The study of alcohols effects on brain explants of the HIPP-ENT area,as an adjunct to in vivo studies, appears not to be a reliable predictor of alcohol-induced neurodegeneration or a system to evaluate possible therapeutic cytoprotective agents. - organotypic culture, explants, neurotoxicity, brain, hippocampus, entorhinal cortex, neurodegeneration, oxidative stress,